Trans-ethnic study confirmed independent associations of HLA-A*02:06 and HLA-B*44:03 with cold medicine-related Stevens-Johnson syndrome with severe ocular surface complications

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Trans-ethnic study confirmed

independent associations of

HLA-A

*

02:06

and

HLA-B

*

44:03

with cold

medicine-related Stevens-Johnson

syndrome with severe ocular surface

complications

Mayumi Ueta

1,2

, Chitra Kannabiran

3

, Tais Hitomi Wakamatsu

4

, Mee Kum Kim

5

, Kyung-Chul Yoon

6

,

Kyoung Yul Seo

7

, Choun-Ki Joo

8

, Virender Sangwan

9

, Varsha Rathi

9

, Sayan Basu

9

, Almas Shamaila

3

,

Hyo Seok Lee

6

, Sangchul Yoon

7

, Chie Sotozono

1

, Jose´ A´lvaro Pereira Gomes

4

, Katsushi Tokunaga

10

& Shigeru Kinoshita

1

1Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan,2Research Center for Inflammation and

Regenerative Medicine, Faculty of Life and Medical Sciences, Doshisha University, Kyoto, Japan,3Prof Brien Holden Eye Research Centre, L V Prasad Eye Institute, Hyderabad, India,4Department of Ophthalmology, Federal University of Sa˜o Paulo, Sa˜o Paulo, Brazil,5Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Korea,6Department of

Ophthalmology, Chonnam National University, Gwangju, Korea,7Department of Ophthalmology, Severance Hospital, Institute of Vision Research, Yonsei University College of Medicine, Seoul, Korea,8Department of Ophthalmology & Visual Science, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea,9Cornea and Anterior Segment Services, L V Prasad Eye Institute, Hyderabad, India,10Department of Human Genetics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Stevens-Johnson syndrome (SJS) and its severe variant, toxic epidermal necrolysis (TEN), are acute

inflammatory vesiculobullous reactions of the skin and mucous membranes. Cold medicines including

non-steroidal anti-inflammatory drugs and multi-ingredient cold medications are reported to be important

inciting drugs. Recently, we reported that cold medicine related SJS/TEN (CM-SJS/TEN) with severe

mucosal involvement including severe ocular surface complications (SOC) is associated with

HLA-A

*

02:06

and

HLA-B

*

44:03 in the Japanese. In this study, to determine whether HLA-B

*

44:03 is a common risk

factor for CM-SJS/TEN with SOC in different ethnic groups we used samples from Indian, Brazilian, and

Korean patients with CM-SJS/TEN with SOC, and investigated the association between CM-SJS/TEN with

SOC and

HLA-B

*

44:03 and/or HLA-A

*

02:06. We found that HLA-B

*

44:03 was significantly associated

with CM-SJS/TEN with SOC in the Indian and Brazilian but not the Korean population, and that

HLA-A

*

02:06 might be weakly associated in the Korean- but not the Indian and Brazilian population.

S

tevens-Johnson syndrome (SJS) and its severe variant, toxic epidermal necrolysis (TEN) with spots, are

acute inflammatory vesiculobullous reactions of the skin and mucous membranes such as the ocular

surface, oral cavity, and genitals. They are rare but often associated with inciting drugs and/or infectious

agents

1–3

.

The association between human leukocyte antigen (HLA) genotypes and drug-induced severe cutaneous

adverse reactions (SCARs) including SJS/TEN has been reported. There was a strong association between

HLA-B

*

58:01

and SCARs, including SJS/TEN and the drug-induced hypersensitivity syndrome (DIHS),

induced by the uric acid lowering drug allopurinol. This association was observed in Han Chinese-

4

,

Caucasian-

5

, and Japanese patients

6

, suggesting that different ethnic groups share the same risk factor(s)

for allopurinol-induced SCARs.

HLA-B

*

15:02

exhibited a very strong association with

carbamazepine-OPEN

SUBJECT AREAS:

GENETICS RESEARCH

RISK FACTORS

Received

2 June 2014

Accepted

15 July 2014

Published

7 August 2014

Correspondence and requests for materials should be addressed to M.U. (mueta@koto. kpu-m.ac.jp)

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induced SJS/TEN in Taiwanese Han Chinese patients

7

and

HLA-A

*

31:01

was strongly associated with carbamazepine-induced

SCARs including SJS/TEN in Japanese-

8

and European patients

9

.

We recently reported that cold medicine-related SJS/TEN with

severe mucosal involvement including severe ocular surface

com-plications (SOC) is associated with

HLA-A

*

02:06

and

HLA-B

*

44:03

in Japanese patients

10

.

The ophthalmologists Mondino et al.

11

and the dermatologists

Roujeau et al.

12,13

reported that

HLA-B12

(HLA-Bw44) was

signifi-cantly increased in Caucasian SJS patients many of whom developed

SJS/TEN after taking non-steroidal anti-inflammatory drugs

(NSAIDs).

HLA-B12

is primarily coded by

HLA-B

*

44:02

or

HLA-B

*

44:03

(http://www.allelefrequencies.net/). The significant

asso-ciation between HLA-B12 and SJS/TEN in Caucasian patients may

be attributable to their genetic background.

To determine whether

HLA-B

*

44:03

is a common risk factor for

CM-SJS/TEN with SOC in different ethnic groups we used samples

from Indian, Brazilian, and Korean patients with CM-SJS/TEN with

SOC, and investigated the association between CM-SJS/TEN with

SOC and

HLA-B

*

44:03

and/or

HLA-A

*

02:06.

Methods

Our study was approved by the institutional review boards of the participating institutions. All experimental procedures were conducted in accordance with the principles of the Helsinki Declaration. The purpose of the research and the experi-mental protocols were explained to all participants, and their prior written informed consent was obtained.

Patients and controls.Ophthalmologists diagnosed SJS/TEN based on a confirmed

history of acute-onset high fever, serious mucocutaneous illness with skin eruptions, and involvement of at least two mucosal sites including the ocular surface1,14,15. They

defined patients with SOC as those who manifested a pseudomembrane and an epithelial defect on the ocular surface in the acute stage, and as patients with ocular sequelae such as dry eye, trichiasis, symblepharon, and conjunctival invasion into the cornea in the chronic stage.

As in our previous study, we focused on SJS/TEN with SOC suspected of having been induced by cold medicines such as multi-ingredient cold medications and NSAIDs. As we found earlier that the genetic predisposition might be different between SJS/TEN with and without severe mucosal involvement including SOC10we

focused on patients from different ethnic groups who presented with SJS/TEN with SOC.

Samples from Indian patients with CM-SJS/TEN were collected at the LV Prasad Eye Institute (n520; 12 males, 8 females; age range 7 to 63 years; median age 27.16

13.4 (SD) years). Their age at onset ranged from 3 to 42 years (median age at onset, 19.2612.2 (SD) years; in 8 patients the age at onset was unknown). The drugs administered to these patients and the HLA type (A and B) of patients with CM-SJS/ TEN with SOC are shown in Supplemental Table 1. The specific drug(s) were not known in all patients. Healthy volunteers (n555; 29 males, 26 females; median age 36.0611.6 years) served as the Indian controls.

Samples from Brazilian patients with CM-SJS/TEN were collected at the Federal University of Sao Paulo (n539, 15 males, 24 females; age range 13 to 69 years; median age, 37.1615.9 years; age range at onset, 3 to 69 years; median age at onset, 24.06

17.2 years). The drugs administered, the ethnicity, and the HLA type (A and B) of these CM-SJS/TEN patients with SOC are shown in Supplemental Table 2. Healthy volunteers (n5134; 55 males, 79 females; median age 41.2612.8 years) were the Brazilian controls (ethnicity: pardo, n566; white, n562; black, n54, Indian plus white, n52).

Samples from Korean patients with CM-SJS/TEN were collected at the Seoul National University College of Medicine, Chonnam National University, Yonsei University, and the Catholic University of Korea. There were 31 patients (12 males, 19 females) ranging in age from 4 to 71 years (median age 33.7619.0 years). Their age at SJS/TEN onset ranged from 3 to 63 years (median age at onset, 23.0616.1 years). The drugs used and the HLA type (A and B) of these patients with SOC are presented in Supplemental Table 3. The specific drug(s) were not known in all patients. Healthy volunteers (n590; 35 males, 55 females; median age 31.767.9 years) were the Korean controls.

Samples from Indian subjects were obtained by extracting DNA from whole peripheral blood with the phenol chloroform method. For Brazilian samples, DNA was extracted from whole peripheral blood using the PAX gene blood DNA kit (Qiagen, Hilden, Germany) or from saliva using Oragene DNA (Kyodou International, Kanagawa, Japan). To obtain the samples from Korean subjects, DNA was extracted from whole peripheral blood using the PAXgene Blood DNA kit (Qiagen).

HLA genotyping.For the analysis ofHLA-AandHLA-Bwe performed polymerase

chain reaction (PCR) assays followed by hybridization with sequence-specific oligonucleotide probes using commercial bead-based typing kits (Wakunaga,

Table

1

|Results

of

association

analyses

in

patients

with

CM-SJS/TEN

with

SOC

ethnic group HLA genotyp e Car rier freq uency (%) Dominant model anal ysis Gene frequ ency (%) Domin ant mod el analysis CM-SJS/T EN with SOC Control P Pc Odds ratio CM-SJS/TEN with SOC Contr ol P Pc Odds ratio (95% CI) (95 % CI) Indian A * 02:06 1/20 (5.0% ) 3/55 (5.5% ) 0.938 -0.9 1 (0.09 –9.31) 1/39 (2.5%) 3/11 0 (2.7% ) 0.9 39 -0.9 1 (0.09 –9.06) B * 44 :03 12/2 0 (60 .0%) 6/55 (10.9%) 1.07 .E-05 2.14. E-05 12.25 (3.57 – 42. 01) 17/40 (42.5%) 7/11 0 (6.4% ) 9. 37.E-08 1. 87.E-07 10.88 (4.04 – 29. 3) Brazilian A * 02:06 0/39 (0.00 %) 0/134 (0.00%) -0/78 (0.00%) 0/26 8 (0.00 %) -B * 44 :03 10/3 9 (25 .6%) 15/134 (11.2%) 0.0239 0 .0478 2.74 (1.12 – 6.7 1) 11/78 (14.1%) 15/2 68 (5.60 %) 0.012 1 0.0242 2.77 (1.22 – 6. 31) Kore an A * 02:06 11/3 1 (35 .5%) 14/90 (15.6%) 0.0181 0 .0362 3.00 (1.18 – 7.5 7) 12/62 (19.4%) 16/1 80 (8.9% ) 0.026 3 0.052 6 2.46 (1.09 – 5. 54) B * 44 :03 6/31 (19 .4%) 18/90 (20.0%) 0.938 -0.9 6 (0.34 –2.69) 7/62 (11.3%) 19/1 80 (10.6 %) 0.8 72 -1.0 7 (0.43 –2.70) P: P values obtained with the x 2-test (Pearson), C I: Confid ence interval. Pc: P values corrected for the multiplic ity of testing by the number of comparisons 2 (HLA-A * 02:06 1 HLA-B * 44:03). CM-SJS/T EN: cold medicine-related S JS/T EN, SOC: severe ocular surfac e complications.

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Hiroshima, Japan). Briefly, the target DNA was PCR-amplified with biotinylated primers specifically designed for amplified exons 2 and 3 of HLA-A, and -B genes. Then the PCR amplicon was denatured and hybridized to complementary oligonucleotide probes (72 probes for HLA-A, 93 probes for HLA-B) immobilized on fluorescent-coded microsphere beads. At the same time, the biotinylated PCR product was labeled with phycoerythrin-conjugated streptavidin and immediately examined with Luminex 100 (Luminex, Austin, TX, USA). Genotype determination and data analysis were performed automatically using the WAKFLOW typing software (Wakunaga, Hiroshima, Japan) according to the manufacturer’s instructions.

Statistical analysis.We compared the carrier frequency and gene frequency of

individual HLA alleles in the patients and controls with thex2-test (Pearson) (JMP

version 11 software; SAS Institute Japan Ltd., Tokyo, Japan).

Results

Strong association between

HLA-B

*

44:03

and CM-SJS/TEN with

SOC in Indian patients

.

We genotyped HLA-A and HLA-B in

samples from Indian subjects (20 CM-SJS/TEN with SOC patients

and 55 controls). Although the number of Indian subjects was small,

we found a strong and significant association between their CM-SJS/

TEN with SOC and

HLA-B

*

44:03

(carrier frequency: p

5

1.07

3

10

25

, odds ratio (OR)

5

12.25, gene frequency: p

5

9.37

3

10

28

, OR

5

10.88) but not

HLA-A

*

0206

(Table 1).

Significant association between

HLA-B

*

44:03

and CM-SJS/TEN

with SOC in Brazilian patients

.

Next we genotyped HLA-A and

HLA-B in samples from Brazilian subjects (39 CM-SJS/TEN with

SOC patients and 134 controls). Although the number of Brazilian

subjects was small we found a significant association between

Brazilian patients with CM-SJS/TEN with SOC and

HLA-B

*

44:03

(carrier frequency: p

5

0.0239, OR

5

2.74, gene frequency: p

5

0.0121, OR

5

2.77) but not

HLA-A

*

0206

which is absent in the

Brazilian population (Table 1). Interestingly, in Caucasians in the

Brazilian samples (Brazilian Caucasian CM-SJS/TEN with SOC

patients: n

5

15, Brazilian Caucasian controls: n

5

62), the

association with

HLA-B

*

44:03

was stronger (carrier frequency:

p

5

0.0037, OR

5

6.22, gene frequency: p

5

0.0011, OR

5

5.99).

Association between

HLA-A

*

02:06

and Korean patients with

CM-SJS/TEN with SOC

.

We also genotyped HLA-A and HLA-B in

samples from Koreans (31 patients with CM-SJS/TEN with SOC

and 90 controls). Although the number of Korean patients was

small we found a significant association between patients with

CM-SJS/TEN with SOC and

HLA-A

*

0206

(carrier frequency: p

5

0.0362, OR

5

3.00, gene frequency: p

5

0.0263, OR

5

2.46) but not

HLA-B

*

44:03

(Table 1).

Discussion

We previously reported that in the Japanese, CM-SJS/TEN with

severe mucosal involvement including SOC was associated with

HLA-A

*

02:06

and

HLA-B

*

44:03

10

. In the present study we

investi-gated whether the association with these alleles is shared by other

ethnic groups. We found that

HLA-B

*

44:03

was strongly associated

with CM-SJS/TEN with SOC in the Indian population which is

gen-etically close to European populations

16

and significantly associated

in the Brazilian population which is comprised of individuals with

different ethnic backgrounds. There was no association between

HLA-B

*

44:03

and CM-SJS/TEN with SOC in the Korean population.

HLA-A

*

02:06

was weakly associated in the Korean population which

is genetically close to the Japanese, but not in the Indian and Brazilian

population.

HLA-B12

(HLA-Bw44) was significantly increased in Caucasian

SJS patients many of whom developed SJS/TEN after taking

NS-AIDs

11–13

. Because

HLA-B12

is primarily coded by

HLA-B

*

44:02

or

HLA-B

*

44:03

(http://www.allelefrequencies.net/), the significant

association of

HLA-B12

with SJS/TEN in Caucasian patients may

be attributable to the association with the

HLA-B

*

44:03

genotype.

We also found that in Brazilian Caucasian patients with CM-SJS/

TEN with SOC, the significant association with

HLA-B

*

44:03

was

stronger than in the entire study population of Brazilians with

CM-SJS/TEN with SOC. To determine whether

HLA-B

*

44:03

is a

com-mon marker for CM-SJS/TEN with SOC in Caucasian, HLA analysis

of European patients with CM-SJS/TEN with SOC is needed.

Although

HLA-A

*

02:06

was strongly associated with the Japanese

CM-SJS/TEN with SOC, and the Korean and Japanese population is

genetically close

16

, in Korean patients CM-SJS/TEN with SOC was

not strongly associated with

HLA-A

*

02:06. To determine whether

HLA-A

*

02:06

is a common marker for CM-SJS/TEN with SOC in

East Asian populations further investigations using a larger number

of samples are needed.

We also performed a meta-analysis by adding our

previously-reported samples

10

. We used Cochran-Mantel-Haenszel statistics

and found that both HLA-A

*

02:06 and HLA-B

*

44:03 are

signifi-cantly associated with CM-SJS/TEN with SOC (Supplemental

Table 4).

SCARs including SJS/TEN and DIHS induced by allopurinol were

commonly and strongly associated with

HLA-B

*

58:01

in patients of

different ethnic backgrounds including Han Chinese-

4

, Caucasian-

5

,

and Japanese patients

6

. This observation suggests that different

eth-nic groups share the same risk factor(s) for allopurinol-induced

SCARs.

With respect to carbamazepine-induced SJS/TEN, different HLA

alleles are associated.

HLA-B

*

15:02

is associated in Taiwanese Han

Chinese patients

7

and

HLA-A

*

31:01

in Japanese-

8

and European

patients

9

.

In CM-SJS/TEN with SOC, the associated alleles we identified are

HLA-A

*

02:06

in Japanese and Korean patients and

HLA-B

*

44:03

in

Indian-, Brazilian-, and Japanese patients. Studies are underway to

determine whether other HLA alleles are associated with CM-SJS/

TEN with SOC in other populations.

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Acknowledgments

This work was conducted as part of the BioBank Japan Project supported by the Ministry of Education, Culture, Sports, Science and Technology of the Japanese government, and as part of the Promotion Project of Knowledge-Based Industrial Clustering of Okinawa Prefecture. It was supported in part by grants-in-aid for scientific research from the Japanese Ministry of Health, Labour and Welfare, a research grant from the Kyoto Foundation for the Promotion of Medical Science, and the Intramural Research Fund of Kyoto Prefectural University of Medicine. The funding agencies had no role in the study design, data collection and analysis, the decision to publish, or the preparation of the manuscript.

Author contributions

M.U. wrote the main manuscript text and prepared the tables. M.U., C.K., T.W., M.K., K.Y., K.S., C.J., V.S., V.R., S.B., A.S., H.L., S.Y., C.S., J.G., K.T. and S.K. contributed to material of the research and reviewed the manuscript.

Additional information

Supplementary informationaccompanies this paper at http://www.nature.com/

scientificreports

Competing financial interests: The authors declare no competing financial interests.

How to cite this article:Ueta, M.et al. Trans-ethnic study confirmed independent

associations ofHLA-A*02:06andHLA-B*44:03with cold medicine-related Stevens-Johnson syndrome with severe ocular surface complications.Sci. Rep.4, 5981; DOI:10.1038/srep05981 (2014).

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder in order to reproduce the material. To view a copy of this license, visit http:// creativecommons.org/licenses/by-nc-nd/4.0/

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